Embryonic stem cells are pluripotent cells derived from the epiblast of the early embryo. In culture they exhibit two important properties: they can maintain an undifferentiated state indefinitely, and they can be pushed to differentiate into a variety of cell types. This proposal targets the molecular dissection of pluripotency and self-renewal in both mouse and human embryonic stem cells (MESCs and HESCs), comparatively. We have recently obtained, and successfully cultured, five of the NIH-approved HESC lines (NIH codes WA01, BG01, BG02, ES03, and ES04). Our preliminary results provide a signature for the state of stemness in both ESCs. We have also demonstrated that the activation of the Wnt pathway is sufficient to maintain the undifferentiated phenotypes in both species. I propose two specific aims: 1- Refinement of the global and comparative molecular profiling of HESCs, and its comparison with MESCs. This allows the establishment of: (i) quantitative molecular standards, for undifferentiated HESCs, (ii) characterization of causal genes for the state of stemness, and (iii) measurement of molecular changes during differentiation paradigms. 2- Characterization of the role of signaling pathways underlying pluripotency, by (i) testing the activity of a novel small compound GSK3[unreadable] inhibitor, BIO, in vivo in mouse embryos; (ii) addressing the role of TGF[unreadable] signaling in this process; and (iii) performing an unbiased expression cloning to identify molecular factors involved in self-renewal in HESCs. In addition to providing a window into early human embryogenesis, HESCs could constitute a renewable source of a large variety of differentiated cells and be employed to replace diseased or damaged tissue by cellular transplantation. Therefore, the establishment of molecular standards and global molecular knowledge of their pluripotency is an obligatory step toward the design of rational clinical treatments. [unreadable] [unreadable] [unreadable]